Molecular Basis of Ecdysteroid Action in the Mosquito

描述（由申请人提供）：在这个项目的前五年，我们对20-羟基蜕皮激素（20E）在蚊子卵黄形成中的作用的分子基础有了基本的了解。本应用程序的目的是进一步表征在成年雌性蚊子中介导20E作用的遗传调控层次。我们克隆了埃及伊蚊20E遗传等级的几个关键因子的同工型。在这个应用中，我们将利用RNAi基因敲除技术，系统地研究每个亚型的作用。在蚊子卵黄发生过程中，受表皮甾体调节级联调控的靶基因的数量和特性尚不清楚。我们将使用基因组微阵列方法结合特定基因的RNAi敲除来描绘新的基因组网络和靶基因。然后，我们将测试多种调控途径与20E调控级联相结合的假设。20E靶效应基因的调控区域，如编码蛋黄蛋白前体的区域，含有EcR/USP和早期基因产物E74、E75和BR （broad complex）的多个结合位点。我们将研究这些因子如何参与调控效应基因的转录活性。我们还将验证这样一个假设，即早期基因产物在与各自的DMA位点结合后，作为转录调节剂直接与EcR/USP相互作用，并以20e依赖的方式共同引发效应基因的高水平表达。我们已经证明，在卵黄形成过程中，能力因子FTZ-F1是激活20E调控层次所必需的。在本应用中，我们将测试FTZ-F1通过招募EcR/USP的共激活子来指导阶段特异性、20e诱导的基因表达的假设。我们发现，USP与抑制因子HR38和SVP或与激活因子EcR的异源二聚化可调节对20E的卵黄反应的循环性。在这个应用程序中，我们将检查EcR是否存在于与热休克蛋白或其他因素的复合体中。这项研究有望显著提高我们对蚊卵发育关键调控级联分子机制的认识，进而可用于开发针对这些最重要的人类疾病媒介的新控制方法。

DESCRIPTION (provided by applicant): During the previous five years of this project, we achieved a fundamental understanding of the molecular basis of 20-hydroxyecdysone (20E) action in mosquito vitellogenesis. The goal of this application is to further characterize the genetic regulatory hierarchy mediating 20E action in the adult mosquito female. We cloned isoforms for several key factors of the 20E genetic hierarchy from Aedes aegypti. In this application, we will take advantage of the RNAi knockout technique, well established for mosquitoes, to systematically study the role of each isoform. Little is known about number and identities of target genes regulated by the ecdysteroid regulatory cascade during mosquito vitellogenesis. We will use the genomic microarray approach in combination with RNAi knockouts of specific genes to delineate novel genomic networks and target genes. We will then test the hypothesis that multiple regulatory pathways are integrated with the 20E regulatory cascade. Regulatory regions of 20E target effector genes, such as those encoding for yolk protein precursor, contain multiple binding sites for EcR/USP and the early gene products E74, E75, and BR (broad complex). We will study how these factors are involved in regulating the transcriptional activity of effector genes. We will also test the hypothesis that the early gene products, upon their binding to respective DMA sites, serve as transcriptional regulators that directly interact with the EcR/USP and cooperatively elicit a high level of expression of an effector gene in a 20E-dependent manner. We have shown that the competence factor FTZ-F1 is required for activation of the 20E regulatory hierarchy during vitellogenesis. In this application, we will test the hypothesis that FTZ-F1 directs stage-specific, 20E-induced gene expression by recruiting co-activator(s) for EcR/USP. We have discovered that alternative heterodimerization of USP either with the repressors HR38 and SVP or with an activator EcR regulates the cyclicity of vitellogenic response to 20E. In this application, we will examine whether EcR exists in a complex with heat shock proteins or other factors during the state of arrest. The proposed research is expected to advance significantly our knowledge concerning molecular mechanisms governing the key regulatory cascade in mosquito egg development, which in turn could be used for developing novel control methods against these most important vectors of human diseases.